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Introduction

Mycobacterium tuberculosis infection is a major health problem that predominantly affects developing countries and is associated with poverty and malnutrition [1, 2]. One-third of the global population is considered to be infected with M. tuberculosis mainly in a latent form which is difficult to be clearly diagnosed which can be reactivated after an alteration of the host immunity. Approximately 5–10% of infected individuals develop an active disease and the majority of healthy individuals are asymptomatic; the infection can remain in a latent form for many years and as long as the immune system can control bacterial dormancy [3]. However, alterations of the host immune system by different causes such as immunodeficiency due to HIV, malnutrition, complications of aging, and some genetic factors can favor a reactivation of latent tuberculosis infection. The use of TNF inhibitors for treatments of severe inflammatory diseases, such as rheumatoid arthritis, Crohn’s disease, and ulcerative colitis, has been associated with reactivation of latent tuberculosis and increased susceptibility to primary tuberculosis infection [4–8]. TNF possesses a broad range of activities required for maintenance of host immunity against mycobacterial infections. A non-redundant activity of TNF involves the development of functional granulomas and the activation of bactericidal mechanisms to control and eliminate intracellular bacilli and maintenance of latent infection. Although the majority of TNF activities have been attributed to the soluble TNF (solTNF) form, the transmembrane or membrane-bound TNF (mTNF) plays an important role in the control of acute tuberculosis infection. Other closely related TNF ligands such as lymphotoxin (LT) α and β play also a role in immunity to mycobacterial infections. This review deals with the protective roles of solTNF and mTNF and other closely related TNF ligands in immunity against mycobacterial infections, and novel therapeutic approaches for selective neutralization of solTNF reducing the risk of infections will be discussed.

Roles of TNF in Mycobacterial Infections

TNF is one of the first cytokines produced by alveolar macrophages after recognition of mycobacteria by Toll-like receptors (TLRs). TNF contributes to the development of granulomas which are highly organized new structures generated by cell-mediated immune responses to mycobacteria that involve the interactions of macrophages, dendritic cells (DCs), giant cells, T cells, B cells, γδT cells, neutrophils, and natural killer (NK) and NK T cells. The granuloma is an area of localized infection characterized by a high cellular activity associated with the production of many cellular mediators including cytokines and chemokines required for cell recruitment, cell circulation to lymph nodes, and development of bactericidal mechanisms. Cytokines and chemokines and the inflammatory response result in the regulated recruitment of inflammatory and immune cells. Bacilli are likely transported by DCs to the lung parenchyma and to draining lymph nodes to prime naïve T cells [9]. CD4 and CD8 T cells expressing IFN-γ, developing T helper 1 (Th1) immunity, and circulating monocytes are recruited to infectious sites and contribute to the granuloma maturation that should contain and destroy some of the bacteria thus preventing dissemination [10]. TNF, mainly released by macrophages and DC, is essential in this coordinated cellular process [11]. A simplified view of how mycobacteria activate cytokine and chemokine production and the important role of TNF in the development of bactericidal mechanisms and cell recruitment to granulomas is depicted in Fig. 20.1. As shown, TNF participates in host resistance in different ways: (1) activating macrophages and monocytes, (2) inducing chemokines and cell recruitment to infected sites, (3) activating bactericidal mechanisms of macrophages, such as the inducible nitric oxide synthase (iNOS or NOS2) producing NO, and apoptosis of infected phagocytes, (4) triggering T cell functions, and (5) synergizing with IFNγ to stimulate anti-mycobacterial mechanisms including autophagy [12].

Fig. 20.1
figure 20_1_183198_1_En

TNF is induced by M. tuberculosis (Mtb) and M. bovis BCG infections. TNF is activated by immune-recognition of mycobacterial through TLRs and NOD2 in macrophages and dendritic cells. Phagocytic cells secrete TNF and other cytokines activating and recruiting monocytes and inducing the development of macrophage bactericidal mechanisms. IFN-γ and TNF are synergistic inducers of microbicidal activities such as NO generation, activation of Irgm involved in autophagy, and apoptosis inhibiting bacillus growth. TNF plays an important role in granuloma formation by participating to immune cell recruitment to infected sites

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Soluble TNF, Membrane-Bound TNF, and Lymphotoxins

TNF is produced as a cell membrane protein and the active form is a trimer. The 26 kDa precursor monomer protein is cleaved into 17 kDa monomers forming biologically active soluble trimers. Proteolytic cleavage is mediated by a cell membrane-bound metalloprotease(s), the TNFα converting enzyme (TACE) [13, 14] that also cleaves other membrane proteins including tumor necrosis factor receptor 2 (TNFR2) [15]. TNF as well as LTα (previously known as TNFβ) are cytokines that provide signals for secondary lymphoid tissue development [16–19]. TNF is produced by monocytes/macrophages, neutrophils, T and B lymphocytes, NK, and many other cells, whereas LTα is secreted by T, B, and NK cells. LTα exists not only as a soluble homotrimer but also in the form of a heterotrimeric membrane molecule in association with a LTβ, forming LTαβ molecules that are found on activated lymphocytes [20, 21]. LTα1β2 is the predominant form, and LTα2β1 is a minor form (<2%) with no defined role [19]. As shown in Fig. 20.2, solTNF and mTNF exert their activities through two receptors which are expressed on the majority of cell types and tissues, 55 kDa TNF receptor 1 (TNFR1) (CD120a) and 75 kDa TNFR2 (CD120b) [22, 23]. LTα binds to TNFR1, to TNFR2, and to another receptor, the herpes virus entry mediator (HVEM). Heterotrimeric LTα1β2, the predominant form, interacts with LTβ receptor (LTβR) which plays an important role in lymph node development, splenic architecture, and lymphoid organ chemokine production required for the recruitment of DCs [24, 25]. LIGHT (lymphotoxin-like, shows inducible expression, competes with HPV glycoprotein D for HVEM, a receptor in T lymphocytes) is a member of the TNF/LT family of ligands, expressed by lymphocytes, monocytes, and DCs, that interacts with LTβR and HVEM and plays a role in co-stimulation of T cells [24, 26, 27].

Fig. 20.2
figure 20_2_183198_1_En

TNF-LT ligands and receptors. Metalloproteases (mainly TACE) cleave the mTNF into the solTNF form (sTNF). TNFR1 and TNFR2 are also released from their membrane into soluble TNFR forms by metalloproteases (open arrows). Interactions between TNF-LT ligands and receptors are indicated by arrows. TNFR1, TNFR2, and LTβR trigger different intracellular signaling including NF-κB, JNK, MAPK, p38MAPK, AP-1, and caspase activation as reviewed [94–96]

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Role of TNF in Experimental Animal Models of Tuberculosis Infection

A critical role of TNF for immunity to mycobacteria has been demonstrated using genetic mouse models of TNF inactivation and pharmacological approaches of TNF inhibition [28]. Several mouse models of infection have contributed to the analysis of the role of TNF during the course of mycobacterial infection. Intravenous infection with the vaccine strain Mycobacterium bovis BCG induces the formation of granulomas in lungs, liver, and spleen, and wild-type C57Bl/6 mice control and survive the infection. However, mice treated with anti-TNF antibodies showed impaired M. bovis BCG granuloma formation and increased bacillus proliferation in infected organs [29]. Sustained inhibition of TNF and LTα by transgenic expression of human TNFR1-Fc fusion protein resulted in fatal infection due to impaired granuloma formation and bacillus overgrowth [30, 31]. Double-deficient TNF/LTα mice were highly susceptible to M. bovis BCG infection, exhibiting a severely impaired immune response with reduced cell recruitment to granulomas and anti-mycobacterial functions [32, 33]. Expression of transgenic LTα in TNF/LTα double-deficient mice prolonged mouse survival after M. bovis BCG infection, showing that the role of TNF is non-redundant for resistance to avirulent mycobacteria. Experimental infections with a virulent M. tuberculosis strain using different routes of infection in mice deficient in TNF, or in mice unable to use TNF by transgenic expression of TNF receptors or by administration of anti-TNF antibodies have shown alteration of granuloma formation associated with extensive lung necrosis and uncontrolled mycobacterial proliferation [30, 34–41]. Using TNF-deficient mice in a model of latent tuberculosis following an antibiotic treatment, arrest of the therapy was followed by a massive reactivation of the disease in TNF deficient mice but not in wild-type mice [42]. The predominant TNF receptor was shown to be TNFR1 for M. bovis BCG, M. tuberculosis, and M. avium resistance [34, 43, 44]. In contrast, TNFR2 appeared to play a minor role in mycobacterial host defense [45] (Fig. 20.2). A study using intra-vital microscopic observation has illustrated the dynamics of macrophages and T-cell interactions during liver M. bovis BCG granuloma formation and disintegration by anti-TNF treatment. This study has confirmed the essential role of TNF for maintenance and functionality of granulomas [46]. Interestingly, a recent study using M. marinum infection in a zebrafish model showed that TNF was not required for granuloma formation but plays an indirect role on granuloma integrity by limiting bacterial growth within macrophages and preventing their necrosis [47].

Membrane TNF Protects from BCG and Acute Tuberculosis Infections

Although the majority of TNF activities were at first attributed to the solTNF form, the specific activities of mTNF have more recently been elucidated. Evidence for a biological role of mTNF has been obtained in vitro as well as in vivo [48, 49–54]. An interesting mode of action of membrane-bound TNF is the induction of reverse signaling, which has been suggested for other members of the superfamily of TNF ligands, although the molecular mechanisms remain uncharacterized [55]. The activation of reverse signaling through mTNF was shown to trigger the protein kinase C pathway and to up-regulate E-selectin on activated human CD4+ T cells [56, 57]. In vitro treatment with anti-TNF antibody activated intracellular signals through mTNF resulting in IL-10 production, cell proliferation, increased apoptosis, and cell cycle arrest of a human T-cell line [58].

In vivo, three different mTNF genetic mouse models have been evaluated for the protective role of mTNF during mycobacterial infections in the absence of solTNF. Olleros et al. investigated the resistance to M. bovis BCG and M. tuberculosis infections in transgenic mice expressing a mTNF (TNFΔ-12-10; Δ-2+1; K11E) under the control of proximal TNF promoter on a TNF/LTα deficient background. Mice were totally resistant to M. bovis BCG infection but only partially protected by mTNF against M. tuberculosis [59, 60]. The protective activity of mTNF has been investigated in two different mTNF knock-in (KI) mouse strains that represent a major advance in terms of regulated expression of mTNF during experimental infection. M. tuberculosis infection of mTNF KI (TNFΔ1-9, K11E) mice showed that mTNF can substitute some of the activities of solTNF during the acute phase of infection but, during the chronic phase of the disease, solTNF appears to be necessary for resistance [61, 62]. M. bovis BCG infection in a second mTNF KI (TNFΔ1-12) mouse strain showed partial control of the infection. These mice also exhibited protection during the acute phase but not in the chronic phase of M. tuberculosis infection whereas TNF-deficient mice rapidly died [63]. These data suggest that mTNF form confers protection during acute phase of M. tuberculosis infection while during the chronic phase solTNF appears to be required for long term immunity.

Lymphotoxins and LIGHT in Immunity to Mycobacterial Infections

The activity of LTα has been closely related to that of TNF since they share the same receptors. Experimental M. bovis BCG and M. tuberculosis infections in LTα KO mice which lack secondary lymphoid organs resulted in uncontrolled bacterial overgrowth, necrotic pulmonary lesions, and animal death [33, 64]. However, a more recently generated LTα KO mouse (neo-free LTα KO), which does not display altered TNF expression [65], called into question the previous studies on LTα KO mice in which TNF expression was decreased by alteration of the TNF locus. Mycobacterial infections of these new LTα KO mice will provide insights on the contribution of LTα in host defense mechanisms (Allie et al, J. Immunol. in press). LTα deficiency involves the lack of LTβ (LTα1β2) and the signaling through LTβR which also plays an important role in macrophage activation. Treatment of M. bovis BCG-infected mice with LTβR-Fc fusion protein, antagonizing LTβ and LIGHT, resulted in reduction of microbicidal macrophage activity and increased bacterial growth [66]. LTβR deficient mice, which lack Peyer’s patches, colon-associated lymphoid tissues, and lymph nodes [67] showed uncontrolled M. tuberculosis infection with a delayed iNOS expression on macrophages forming granulomas [68]. In contrast, LIGHT deficient mice were not more sensitive to M. tuberculosis than wild-type mice indicating that direct cell contact interactions between lymphocytes bearing LTβ and monocytes/macrophages expressing LTβR are critical for the control of M. tuberculosis infection [68].

Neutralization of TNF and Risks of New Infection and Reactivation of Latent Tuberculosis

The large number of patients treated with TNF inhibitors has revealed an increased risk for opportunistic infections, most notably primary infection by M. tuberculosis as well as reactivation of latent tuberculosis which represent an important complication of such treatment [4–8, 69]. It has been shown that anti-TNF therapy decreases the frequency of CD4 T lymphocytes capable of producing IFN-γ upon antigenic activation. These effects can partially explain the increased incidence of tuberculosis in patients treated with TNF inhibitors [70]. One study provides evidence that treatment of rheumatoid arthritis patients with etanercept impaired B cell function by reducing follicular dendritic networks, germinal center structures, and peripheral blood memory B cells which are not affected in patients treated with methotrexate [71]. A recent study has shown that anti-TNF antibodies reduced the population of CD8 effector memory T cells which mediate antimicrobial activity against M. tuberculosis by the expression of granulysin [72]. Many clinical studies are available today showing the association of anti-TNF therapies and the increased risk of primary and reactivated of tuberculosis. Clinical studies also point out that such complications may occur even after chemoprophylaxis for latent tuberculosis.

Strategies to Block TNF Activities

The extension of anti-TNF for use in the treatment of human inflammatory diseases other than rheumatoid arthritis, Crohn’s, and psoriasis also increases the potential for complications associated with these therapies. Thus, new therapeutic strategies are required to attenuate the deleterious effects of total TNF blockade on the host immune system while maintaining the positive anti-inflammatory effects of TNF therapies. Given that this nonselective anti-TNF therapy has proven highly efficacious for severe inflammatory disease, the development of selective inhibitors of solTNF may therefore represent a promising future therapeutic strategy. A novel class of TNF inhibitors is available, known as dominant-negative (DN) TNF, that antagonize solTNF but not mTNF [73]. Recent studies have shown that these DN-TNF biologics are effective in reducing inflammation in mouse arthritis and Parkinson’s disease models, yet in contrast to nonselective inhibition, do not suppress the resistance of mice to Listeria monocytogenes infection [74, 75]. We have analyzed the effects of a DN-TNF on host defense to M. tuberculosis and M. bovis BCG infections, and on protection against endotoxin-induced liver inflammation in M. bovis BCG-infected mice to study their influence during mycobacterial infections as well as their efficacy in preventing an acute inflammatory reaction. DN-TNF efficiently protected from endotoxin-mediated hepatotoxicity in M. bovis BCG-infected mice, while immunity against M. tuberculosis and M. bovis BCG infections was preserved, presumably by maintenance of physiological mTNF signaling [76]. Although long-term studies and murine latent tuberculosis are still required to better evaluate the effect of DN-TNF molecules, selective inhibition of solTNF seems to better preserve immune defenses compared with neutralization of both solTNF and mTNF. The different mechanisms of action of DN-TNF, etanercept, and anti-TNF antibodies are illustrated in Fig. 20.3 which shows that DN-TNF only inhibits solTNF signaling by subunit exchange with endogenous TNF, etanercept interacts with both solTNF and mTNF as well as with LTα and the minor form of LTαβ [77], and anti-TNF antibodies block mTNF and solTNF.

Fig. 20.3
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Mechanisms of action of selective and non-selective TNF inhibitors. Left, DN-TNF, a mutated form of human solTNF (sTNF) with disrupted receptor binding interfaces, eliminates solTNF by a subunit exchange mechanism, but is unable to interact with mTNF, LTα, and LTα2β1. Center, solTNF, mTNF, LTα, and LTα2β1 (the minor form) can be neutralized by soluble TNFR2-Fc (e.g., etanercept) inhibiting interaction with corresponding receptors. Right, anti-TNF (e.g., infliximab, adalimumab, and golimumab) inhibits solTNF and mTNF. DN-TNF biologics inhibit solTNF receptor signaling mediating inflammation without suppressing mTNF and LTα protective immune responses through interaction with TNFR1 and TNFR2

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A study using a TNF virus-like particle-based vaccine that selectively targets solTNF has also shown protection from arthritis without inducing reactivation of latent tuberculosis [78]. Animals immunized with total TNF virus-like particles were highly sensitive to M. tuberculosis infection whereas mice producing antibodies against the N-terminus of TNF were not sensitive to the infection but were protected from arthritis. These differences were attributed to antibodies recognizing both mTNF and solTNF or interacting only with solTNF [78]. These data indicate that selective inhibition of solTNF may be a promising anti-inflammatory approach which does not suppress the immune response to acute or chronic tuberculosis infections.

New promising strategies of TNF inhibition designed at the level of both the TNFR and TNF molecules are being explored today. TNFR1 and TNFR2 contain an extracellular pre-ligand binding assembly domain (PLAD) which is distinct from the ligand binding domain. This region is necessary for the assembly of TNFR complexes in the absence of ligand and encourages trimerization upon activation by TNF [79]. A soluble form of PLAD derived from TNFR1 was shown to block TNF in vitro and inhibit arthritis in animal models [80]. Several human TNF variants with mutations corresponding to the six amino acid residues at the receptor binding site have been selected on the basis of high affinity for TNFR1 and absence of biological activity [81]. One of the TNF mutants showed inhibition of TNFR1 but not TNFR2 activities. This TNF mutant was able to protect mice in several hepatitis models including TNF/D-GalN and ConA [82, 83]. This represents a new TNF inhibitor to be explored for its effects on infectious diseases. Finally, TNF inhibition by small-molecules has been reported [84]. This small molecule inhibited TNF–TNFR interaction by binding to intact biologically active TNF trimers and promoted subunit disassembly thereby inactivating the cytokine in vitro [84].

TNF and Chemokines in Mycobacterial Infections

Mycobacterial infections induce chemokine activation involved in migration of immune cells to the site of the infection thus favoring the development of granulomas [11, 85]. Chemokines are detected in bronchoalveolar lavage (BAL), lymph nodes, and blood from patients with active tuberculosis as reviewed [86].

M. tuberculosis infection of mouse macrophages activates chemokines such as MCP-1/CCL2, MIP-1α/CCL3, and MIP-1β/CCL4 and chemokine receptors CXCR3, CCR5, and CCR2 [87, 88, 89]. Deficiency in CCR2 (the receptor for MCP-1, MCP-3, and MCP-4) has confirmed its requirement for host defense against M. tuberculosis infection, as the majority of CCR2 deficient mice died early after infection and showed severe alteration of cell recruitment in infected lung [90]. In contrast, M. tuberculosis-infected CCR5 (the receptor for RANTES, MIP-1α, and MIP-1β) deficient mice were able to induce Th1 immune responses and to control the infection [91]. Surprisingly, mice deficient in CXCR3 (the receptor for IP-10, ITAC, and Mig) were found to be more resistant to M. tuberculosis than wild-type mice, suggesting that CXCR3 can attenuate the host immune response [92].

Deficiency of TNF during infection affects the recruitment of inflammatory and immune cells to granulomas in mouse models [36]. Studies in TNF-deficient mice showed that RANTES/CCL5, MCP-1/CCL2, and MIP-1β/CCL4 mRNA levels were reduced in TNF KO mice at early infection but were highly increased at 4 weeks of infection [37]. Dysregulation of circulating and pulmonary RANTES and MIP-1α was observed in TNF/LTα KO mice infected with M. bovis BCG [60]. Deficiency in TNF led to in vitro and in vivo reduced and/or delayed production of chemokines by pulmonary macrophages and aberrant granuloma formation [93]. Mice expressing a transgenic mTNF in a TNF/LTα KO background were able to activate RANTES and MCP-1α and developed bactericidal granulomas after M. bovis BCG infection [60]. These data suggest that solTNF and also mTNF play a role in the chemokine gradient of infected tissues determining efficient cell migration and control of infection.

Conclusion

TNF has an essential protective activity in latent and active tuberculosis infection in humans. Neutralization of TNF can have consequences on a non-diagnosed latent infection which can be reactivated and followed by an acute tuberculosis infection. In addition, TNF inhibition renders patients more sensitive to new infections. Genetic and pharmacological animal data showing that inhibition of solTNF sparing mTNF is effective in abrogating inflammatory diseases and yet maintaining host defense activity may allow the improvement of anti-TNF therapy. These data suggest that the risks associated with total TNF inhibition might be reduced by the use of new compounds only blocking solTNF but sparing the protective effects of mTNF. A reduction in the infection risks associated with current anti-TNF drugs may allow the safer use of anti-TNF therapies in other inflammatory diseases.